Calcium dihydroxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 25, 2007 - June 28, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD 201.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

No pH adjustment in test medium.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

No pH adjustment in test medium.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Start test: duplicate samples of all solutions
End test: test solutions of 2 replicates were mixed (precipitate left behind). Additionally, the test solutions of two replicates of the 235 mg/L concentration were mixed with 1N HCL (0.625 ml/replicate) to redissolve any precipitated calcium. Duplicate samples were taken from controls and treated vessels, and from test vessels without algae at the highest test item concentration.
Storage samples: in brown glass bottles at ambient temperature in the dark.

Vehicle:

no

Details on test solutions:

Test item was weighed separately into a glass beaker. Afterwards the content of the respective beakers was transferred to a 1000 mL measuring flask and filled up with temperature adjusted test medium. Turbidity was noted at 138 mg/L and higher, so the test item was not completely dissolved at these concentrations. It is also possible that due to reaction of the test item with test medium components and CO2, poorly soluble precipitates were formed.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata (SAG 61.81) supplied by Institut für Pflanzenphysiologie, University Göttingen, D-37073 Göttingen.
Age of the pre-culture: 3 days
Number of cells per ml in the pre-culture before inoculating the test solution: 1515000
Number of cells per ml test solution at the beginning of the test: 5000
Algal medium pH 5.8

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

107 mg CaCO3/L

Test temperature:

22.2-23.9 degC

pH:

INITIAL:
control: 5.8
48.0 mg/l: 6.4
80.0 mg/l: 6.8
138.0 mg/l: 7.2
235.0 mg/l: 8.1
400.0 mg/l: 11.4

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 0 (control), 48.0, 80.0, 138.0, 235.0 and 400.0 mg/l.
Nominal and measured concentrations were approximately similar.

Details on test conditions:

Test vessels: 300 ml Erlenmeyer flasks covered by air-permeable stoppers
Test medium: Kuhl and Lorenzen (1964) algal medium
Amount of test solution per test vessel: 100 ml
Number of replicates per test item concentration: 3
Number of replicates in the control: 6
Number of replicates for stability check (highest concentration without algae): 2
Light cycle: 24/0 hours light/dark
Type of light: fluorescent tubes of universal white type (L58W/840)
Light intensity: mean value of six measurements: 8025 lx (equivalent to 4440 - 8880 lx)
Temperature: 22.8 ± 0.30 °C
Shaker: 100 ± 5 oscillations/min (the test vessels were placed randomly on the shaker)
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 250 µl of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA). The results were converted into biomass concentration using a calibration curve.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

79.22 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ca(OH)2

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

106.02 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ca(OH)2

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

184.57 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ca(OH)2

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ca(OH)2

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ca(OH)2

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Flocculation: with increasing concentrations precipitates formed over time to which algae adhered, leading to flocculation. Visible precipitates formed at 138 mg/L and above leading to only a marginal increase in pH. It was therefore concluded that the initial pH of the test medium was not directly related to the biologically relevant effects. The formation of precipitates is likely the result of the reaction between Ca(OH)2 and CO2 dissolved in the medium yielding poorly soluble CaCO3.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the measured Ca concentrations were much below the nominal concentrations, due to the reaction of the test item with CO2 to poorly soluble CaCO3, thus forming precipitates. However, measurement of Ca after acidification at the end of the test resulted in a recovery of 97.7% .

Results with reference substance (positive control):

Growth rate: EC50 (0-72h): 1.635 mg/L

Reported statistics and error estimates:

The biological results were evaluated statistically. Probit analysis was used to calculate EC values.

Validity criteria fulfilled:

yes

Remarks:

Mean biomass increase in the control cultures: 139,4. Mean coefficient of variation for section-by-section specific growth rates in the control cultures: 7,0%. Coefficient of variation of average specific growth rates during test period in replicate contr

Conclusions:

A clear concentration-response relationship was observed.
The pH of the medium at concentrations resulting in a considerable growth inhibition, was below 8 and the biological findings are therefore not attributed to the initial pH of the test solutions.
It was observed that, however, that with increasing test item concentrations precipitates were formed over time to which algae adhered, leading to their flocculation. The flocculation of algae is thus considered to be the predominant biologically relevant effect in this system test.
The recovery of the test item at the end of the test was below 80% of the nominal concentration. This can be explained since the test item is known to react with CO2 to calcium carbonate, which is poorly soluble in water leading to the formation of precipitates. However, after acidification, the test item recovery was 97,7% of the nominal concentration, which conforms the establishment of the target concentration

Description of key information

Klimisch 1 study ): growth rate of Pseudokirchneriella subcapitata: nominal EC50(72h) = 184.57 mg Ca(OH)2/L , nominal NOEC(72h) = 48 mg Ca(OH)2/L. (Egeler et al. (2007
Klimisch 1 study; 72-h ErC50 > 14 mg/L  (measured, 100 %v/v saturated solution nominal); NOErC was 14 mg/L (measured, 100 %v/v saturated solution nominal) (Vryenhoef and Mullee, 2010)

Key value for chemical safety assessment

EC50 for freshwater algae:

184.57 mg/L

EC10 or NOEC for freshwater algae:

48 mg/L

Additional information

The acute toxicity to algae of calcium dihydroxide was assessed in a study performed according to OECD TG 201 under GLP (Egeler et al, 2007). In this study Pseudokirchneriella subcapitata were exposed to calcium dihydroxide at nominal concentrations of 0, 48, 80, 138, 235 and 400 mg/L for 72 hours. A clear concentration-response relationship was observed. The pH of the medium at concentrations resulting in a considerable growth inhibition, was below 8 and the biological findings are therefore not attributed to the initial pH of the test solutions. It was observed that, however, that with increasing test item concentrations precipitates were formed over time to which algae adhered, leading to their flocculation. The flocculation of algae is thus considered to be the predominant biologically relevant effect in this system test. The recovery of the test item at the end of the test was below 80% of the nominal concentration. This can be explained since the test item is known to react with CO2 to calcium carbonate, which is poorly soluble in water leading to the formation of precipitates. However, after acidification, the test item recovery was 97.7% of the nominal concentration, which conforms the establishment of the target concentration. The 72-h ErC50 was 184.57 mg/L and the 72-h NOErC was 48 mg/L.

The acute toxicity to algae of calcium carbonate (nano) was assessed in a study performed according to OECD TG 201 under GLP (Vryenhoef and Mullee, 2010). In this study Desmodesmus subspicatus (green algae) were exposed to a 100 %v/v saturated solution of calcium carbonate (measured concentration = 14mg/L). No toxic effects were noted at the concentration tested. Hence, the 72 h ErC50 for calcium carbonate (nano) was found to be >14 mg/L (measured, 100 %v/v saturated solution nominal) and the NOErC was 14 mg/L (measured, 100 %v/v saturated solution nominal). The concentration of calcium carbonate (nano) that might cause acute toxicity is therefore greater than the maximum solubility of calcium carbonate in water.

Based on the results of the studies performed on calcium dihydroxide and calcium carbonate, it may be concluded that the acute toxicity to algae of grades of calcium dihydroxide containing up to 35% calcium carbonate will be driven by the calcium dihydroxide content and that the results available for the calcium dihydroxide itself represent the worse-case for all grades.